Cysteine proteases and peptidases are defined by their use of an active-site cysteine as a nucleophile. Cysteine proteases are divided into clans (proteins which are evolutionary related), and further sub-divided into families, on the basis of the architecture of their catalytic dyad or triad. Barrett and Rawlings, Biol. Chem. 382:727-733, 2001. Members of the papain family of cysteine proteases display a wide variety of activities, including broad-range and narrow-range endopeptidases, aminopeptidases, dipeptidyl peptidases, and enzymes with both exo- and endo-peptidase activity. Members of this family are widespread and may be found in baculovirus, eubacteria, yeast, and practically all protozoa, plants, and mammals. The enzymes are typically lysosomal or secreted. They are often synthesized as inactive proenzymes with N-terminal propeptide regions that are proteolytically cleaved to activate the enzymatic activity.
Examples of enzymes belonging to the papain family of cysteine proteases include papain, bromelain, bleomycin hydrolase, and the cathepsins. There are at least 11 cathepsins encoded in the human genome and 19 in mouse, and each of these proteins displays different expression patterns, levels, and specificities. Turk et al., Cancer Cell 5:409-410, 2004. Several of these proteases are key players in normal physiological processes such as antigen presentation (Villadangos et al, Immun. Rev. 172:109-120, 1999), bone remodeling (Gelb et al., Science 273:1236-1238, 1996), and prohormone processing (Beinfeld, Endocrine 8:1-5, 1998). In addition, several of these proteases are involved in pathological processes such as rheumatoid arthritis (Iwata et al., Arthritis and Rheumatism 40:499-509, 1997), cancer invasion and metastasis (Yan et al., Biol. Chem. 379: 113-123, 1998; Joyce et al., Cancer Cell 5:443-453, 2004), and Alzheimer's disease (Golde et al., Science 255:728-730, 1992; Munger et al., Biochem. J. 311:299-305, 1995).
The enzymatic mechanism used by the papain family of cysteine proteases has been well studied and is highly conserved. Thus, electrophilic substrate analogs that are only reactive in the context of this conserved active site can be used as general probes of function. A wide range of electrophiles have been developed as mechanism-based, cysteine protease inhibitors, including diazomethyl ketones (Shaw, Meth. Enzymol. 244:649-656, 1986), fluoromethyl ketones (Shaw et al. Biomedica Biochimica Acta 45:1397-1403, 1986), acyloxymethyl ketones (Pliura et al., Biochem. J. 288:759-762, 1992), O-acylhydroxylamines (Brömme et al., Biochem. J. 263:861-866, 1989), and vinyl sulfones (Palmer et al, J. Med. Chem. 38:3193-3196, 1995). These inhibitors typically consist of a peptide specificity determinant attached to an electrophile that irreversibly alkylates the enzyme when bound in close proximity to an attacking nucleophile.
A naturally-occurring epoxysuccinyl peptide capable of irreversibly inhibiting cysteine proteases, including cathepsins B, H, and L, was isolated from Aspergillus japonicus in the late 1970s. Barrett et al., Biochem. J. 201:189-198, 1982.
